1. Field of the Invention
The present invention is directed to pH dyes containing a suitable compatible substituent which permits binding to a solid support as well as to solid supports containing such bound dyes. The present invention is also directed to methods for synthesizing such dyes prior to their coupling to the solid supports.
2. State of the Art
The binding of biologically or chemically active components to solid supports is well known in the art. For example, in the field of medical diagnostics, it is common to bind a biologically active component (e.g., an antigen or an antibody) to a solid support for use in a diagnostic assay. The binding of such components facilitates reagent interaction and/or separation.
In the field of analytical chemistry, the binding of a chemically active component to a solid support such as a probe has been suggested as a means of permitting the solid phase incorporation of the component. Such solid phase incorporation eliminates the possibility that the component will leach from the probe when contacted with the intended environment. In this regard, Posch et al., Fresenius Z Anal. Chem., Vol. 334, pp. 162-165 (1989), disclose the binding of fluorescent compounds such as fluorescein to solid supports useful in fibre optics. The fluorescent compound is used in a method to determine the pH of the a solution between the pHs of 0 to 7.
The binding of a fluorescent component, such as fluorescein, rhodamine, etc., to a solid support is facilitated because such components contain one or more reactive functionalities (e.g., amine, hydroxyl, or carboxyl functionalities) which permit the covalent linking of these compounds to a solid support. However, the use of a solid support containing a fluorescent component in fibre optics is complicated by the fact that while most commercially available fibre optics poorly transmit short wavelength light, such short wavelength light is generally employed to excite the fluorescent moiety. Moreover, fluorescent methods are further complicated by problems of quenching and dye photodegradation. Additionally, problems can arise because the binding of the fluorescent component to the solid support can result in significant chemical changes in the bound fluorescent component as compared to the unbound component and these changes can interfere with the intended use of the component. For example, Posch et al., supra, states that upon binding of Rhodamine B to the solid support, there was a strong pK.sub.a shift in the bound component as compared to the unbound component.
On the other hand, certain phthalein type dyes are well known pH indicators. When incorporated into an aqueous solution, these dyes provide for protonated or unprotonated species and depending on the degree of protonation, will produce solutions of different colors or color intensity. When the pH value of a solution produces a color in the presence of the pH indicator, the extent of protonation of the indicator and hence the pH of the solution can be correlated to the intensity of the color of the solution.
However, in solid phase pH sensors, the use of such well known pH dyes as substitutes for fluorescent indicators has not been heretofore possible because such dyes lack suitable functionality which would permit the covalent binding of the dye to the solid support without interfering with the pH sensitivity of the dye. Moreover, it is uncertain as to whether the incorporation of such functionality into the dye would not alter the chemical properties of the dye.